Cdc-42 and the Exocyst in Ciliogenesis and Polycystic Kidney Disease

 描述(由申请人提供)： 囊发生对早期肾脏发育至关重要；然而，如果肾囊变延迟和/或发生在不正确的环境中，所产生的囊变可破坏肾脏结构，从而促进多囊肾病(PKD)和肾功能衰竭。在人类患者和动物模型中，几乎所有形式的PKD都与肾脏初级纤毛结构和/或功能的扰动有关。例如，编码纤毛蛋白Polycystins-1和-2的基因突变会导致常染色体显性遗传性PKD(ADPKD)，这是人类最常见的潜在致命性单基因疾病，而多个纤毛蛋白的突变会导致肾病，这是PKD的一种形式，是儿童和年轻人肾功能衰竭的最常见遗传原因。虽然维持原生纤毛结构和功能似乎是预防/治疗PKD的关键，但纤毛发生异常如何促进疾病尚不完全清楚。此外，由于任何形式的PKD都没有被批准的治疗方法，即使是最有希望的治疗方法也有显著的副作用和/或毒性，因此对初级纤毛形成/功能的更彻底的了解对于确定未来治疗PKD的靶点至关重要。我们实验室的长期目标是确定纤毛发生的机制基础，并确定这一过程中的异常如何促进PKD。我们的实验室是第一个证明高度保守的八蛋白外囊复合体，它促进携带来自跨高尔基网络(TGN)的蛋白质的囊泡的靶向和对接，定位于初级纤毛，并且是形成初级纤毛所必需的。值得注意的是，在一个肾炎家族中发现的胞囊蛋白突变支持了胞囊在预防PKD中的作用。外囊被证明是将带有纤毛蛋白的Rab8阳性囊泡靶向新生纤毛的关键。最近，我们证明了小GTP酶Cdc42和Tuba，一种CDc42纤毛特异性的鸟嘌呤核苷酸交换因子(Global)，都调节了胞囊本身对新生纤毛的靶向，在那里，胞囊的Sec10成分直接与PAR复合体相互作用。最值得注意的是，我们最近证明了斑马鱼中的CDC42基因敲除会导致纤毛缺陷，而小鼠中具有肾脏特异性的CDC42基因敲除会阻止纤毛的发生，这会导致PKD肾病表型、肾功能衰竭和死亡。初步数据显示，在斑马鱼中，Tuba基因敲除会导致类似的表型。基于这些数据，我们假设cdc42被一个或多个纤毛特异的GEF激活，以将胞囊招募到新生纤毛，从而 促进Sec10/Par6稳定的相互作用，允许靶向带有纤毛蛋白的Rab8阳性小泡以促进纤毛发生(在AIMS 1和2中进行了测试)。我们处于进行这些实验的独特地位，因为我们拥有所有必要的工具、技术和试剂。由于正确的纤毛形成和功能对于预防PKD至关重要，进一步了解纤毛蛋白的主要传递途径可能会识别出许多新的 治疗干预的候选靶点和要点。我们还发现，在体外和体内破坏CDC42和Sec10会导致MAPK激活和磷酸化ERK(PERK)增加。由于MAPK的激活发生在肾小球肾炎小鼠身上，而PERK的药物正常化可以防止囊变发生，我们进一步假设，抑制新生纤毛上的胞囊定位/稳定会导致MAPK的异常激活，进而， 通向PKD。在目标3中，我们将采用药理学方法在我们的CDC42和Sec10基因敲除斑马鱼和条件性小鼠基因敲除模型中测试这一假说，并将开发我们最近描述的新腺相关病毒(AAV)载体和逆行递送系统，允许基因在小鼠肾小管细胞中表达。综上所述，我们的工作为PKD的治疗开辟了新的途径：利用药物手段抑制MAPK途径，利用分子生物学传递野生型基因来挽救遗传性纤毛疾病。

 DESCRIPTION (provided by applicant): Cystogenesis is critical for early kidney development; however, if renal cystogenesis is delayed and/or occurs in an incorrect context, the resulting cysts can destroy kidney structure, thereby promoting polycystic kidney disease (PKD) and renal failure. Virtually all forms of PKD in human patients and animal models are associated with perturbations in renal primary cilia structure and/or function. For example, mutations in genes encoding the ciliary proteins polycystins-1 and -2 cause autosomal dominant PKD (ADPKD), the most common potentially lethal monogenic disorder in humans, while mutations in multiple ciliary proteins lead to nephronophthisis, a form of PKD that is the most common genetic cause of renal failure in children and young adults. Although maintenance of primary cilia structure and function appears to be key to preventing/treating PKD, it is not entirely clear how aberrant ciliogenesis promotes disease. Moreover, since there are no approved treatments for any form of PKD, and even the most promising treatments have significant side effects and/or toxicities, a more thorough understanding of primary cilia formation/function is critical for the identification of future therapeutic targets for the treatment of PKD. The long-term goal of our laboratory is to identify the mechanistic basis for ciliogenesis, and determine how aberrations in this process contribute to PKD. Our laboratory was the first to show that the highly-conserved eight-protein exocyst complex, which promotes targeting and docking of vesicles carrying proteins from the trans-Golgi network (TGN), localizes to, and is necessary for formation of, primary cilia. Notably, mutations in an exocyst protein, identified in a family with nephronophthisis, support a role for the exocyst in prevention of PKD. The exocyst has been shown to be critical for targeting Rab8-positive vesicles bearing ciliary proteins to the nascent cilium. More recently, we demonstrated that both the small GTPase Cdc42 and Tuba, a Cdc42 ciliary-specific guanine nucleotide exchange factor (GEF), regulate targeting of the exocyst itself to the nascent cilium, where the Sec10 component of the exocyst directly interacts with the Par complex. Most notably, we recently demonstrated that cdc42 knockdown in zebrafish causes ciliary defects and Cdc42 kidney-specific knockout in mice prevents ciliogenesis, which leads to a PKD nephronophthisis phenotype, renal failure, and death. Preliminary data show that tuba knockdown in zebrafish results in a similar phenotype. Based on these data, we hypothesize that activation of Cdc42 by one or more ciliary-specific GEFs is required to recruit the exocyst to the nascent cilium, thereby promoting Sec10/Par6 stabilizing interactions that allow for the targeting of Rab8-positive vesicles bearing ciliary proteins to promote ciliogenesis (tested in Aims 1 and 2). We are in a unique position to undertake these experiments as we have all the necessary tools, techniques, and reagents. As proper cilia formation and function are critical for preventing PKD, further understanding of a major delivery route for ciliary proteins will likely identify a number of novel candidate targets and points of therapeutic intervention. We also showed that Cdc42 and Sec10 disruption in vitro and in vivo leads to MAPK activation and an increase in phosphorylated ERK (pERK). As MAPK activation occurs in the nephronophthisis pcy mouse, and pharmacologic normalization of pERK prevents cystogenesis, we further hypothesize that inhibition of exocyst localization/stabilization at the nascent cilium results in abnormal MAPK activation that, in turn, leads to PKD. In Aim 3, we will take a pharmacologic approach to test this hypothesis in our Cdc42 and Sec10 knockdown zebrafish and conditional murine knockout models, and will also exploit our recently described novel adeno-associated viral (AAV) vector, and retrograde delivery system, that allows for gene expression in murine kidney tubule cells. Taken together, our work opens up new avenues for the treatment of PKD: utilizing pharmacologic means to inhibit the MAPK pathway, and molecular biology to deliver wild-type genes to rescue inherited ciliopathies.